Identification of serine hydrolases involved in Plasmodium host cell invasion

描述（由申请人提供）：恶性疟原虫是导致疟疾的寄生虫，这种疾病继续在世界所有热带地区肆虐。每年报告的疟疾病例有25万例，其中近100万例导致死亡。控制这种疾病的努力由于寄生虫不断增长的耐药性而变得复杂，导致迫切需要新的药物靶点。鉴定新靶点的一个途径是通过用小分子抑制寄生虫中的基本酶来发现和表征它们。丝氨酸水解酶是一个大家族的酶，具有高活性和共价修饰的活性位点，使它们具有小分子抑制的吸引力。此外，丝氨酸水解酶在包括人类在内的大多数生物中都有很好的表现，它们占所有蛋白质的1%。在恶性疟原虫中已经鉴定出两种必需的丝氨酸水解酶，枯草杆菌样丝氨酸蛋白酶1 （PfSUB1）和2 （PfSUB2）。为了在疟原虫中发现其他必需丝氨酸水解酶，在哺乳动物中筛选了高效丝氨酸水解酶抑制剂文库。这个三唑脲化合物文库揭示了几种减缓或阻断恶性疟原虫生长的化合物。最有效的化合物AA691似乎能诱导宿主细胞入侵缺陷。此外，在竞争分析中已经证实AA691与氟膦酸盐（FP）（一种通用的丝氨酸水解酶抑制剂）结合至少一个相同的靶标，这表明这种缺陷是由于丝氨酸水解酶的抑制。本项目将利用基于共价修饰其靶标的AA691结构的探针分离受AA691抑制的丝氨酸水解酶。然后将使用串联正交蛋白水解-基于活性的蛋白谱（TOP-ABPP）和质谱法鉴定目标。这些靶标在宿主细胞侵袭中的作用将在培养中通过在恶性疟原虫中创建直接和可调节的条件敲除来表征。侵袭性缺陷将在体内用柏氏疟原虫小鼠感染进一步验证。最后通过肝细胞培养和体内实验，评价AA691对肝分期的影响。间日疟原虫感染在肝脏中处于休眠状态，导致血液期治疗后复发，其流行率不断上升，这推动了对肝期寄生虫的治疗。迫切需要新的疟疾治疗方法，而这项研究的结果可能直接导致这些药物的开发。

DESCRIPTION (provided by applicant): Plasmodium falciparum is the parasite responsible for malaria, a disease that continues to devastate all tropical regions of the world. A quarter of a million cases of malaria are reported annually with close to a million of these cases resulting in death. Efforts to control this disease are complicated by growing drug resistance in the parasite, leading to an imminent need for new drug targets. One avenue to the identification of novel targets is to discover and characterize essential enzymes in the parasite by inhibiting them with small molecules. The serine hydrolases are a large family of enzymes with highly reactive and covalently modifiable active sites making them attractive for inhibition with small molecules. Furthermore serine hydrolases are well represented in most organisms including humans where they comprise 1% of all proteins. In P. falciparum two essential serine hydrolases have already been identified, subtilisin-like serine protease 1 (PfSUB1) and 2 (PfSUB2). In order to find additional essential serine hydrolases in the parasite, a library of highly effective serine hydrolase inhibitors in mammals was screened in P. falciparum. This library of triazole urea compounds revealed several compounds that slow or block the growth of P. falciparum. The most efficacious compound, AA691, appears to induce a host cell invasion defect. Furthermore it has been verified that AA691 binds to at least one of the same targets as fluorophosphonate (FP), a generic serine hydrolase inhibitor, in competition assays suggesting this defect is due to inhibition of serine hydrolases. In this project the serine hydrolases inhibited by AA691 will be isolated using a probe based on the structure of AA691 that covalently modifies its targets. The targets will then be identified using tandem orthogonal proteolysis-activity-based protein profilin (TOP-ABPP) and mass spectrometry. The role(s) of these targets in host cell invasion will be characterized in culture through the creation of direct and regulatable conditional knockouts in P. falciparum. The invasion defect will be further verified in vivo with P. berghei mouse infections. Finally the effects of AA691 on the liver stage will be assessed in hepatocyte culture and in vivo. The rising prevalence of P. vivax infection, which remains dormant in the liver leading to relapse after blood stage treatment, is driving a push for therapeutics against liver stage parasites. There is an urgent need for novel malaria therapeutics and the results of this research could lead directly to the development of these drugs.